Pandey, Shashank - Decoding the molecular control of bud dormancy release in trees

Research


Shashank Pandey is standing in a greenhouse with small aspen trees on a table in front of him and taller ones behind him, he is looking straight in the cameraPhoto: Jasim Basheer

The DECORE project aims to uncover mechanisms by which low temperature controls bud dormancy in hybrid aspen. Recent research in our lab identified AGL8 as a transcription factor involved in the release of bud dormancy. Building on this, we hypothesize that AGL8 regulates gibberellin, the mobile signaling component FT1, and intercellular communication to coordinate the release of bud dormancy.

Plants have an astonishing ability to sense and respond to environmental cues, including seasonal changes. One critical but poorly understood aspect of this responsiveness is the regulation of bud dormancy in trees, a phenomenon central to their survival and growth in temperate regions. My MSCA research, focusing on the experimental model tree hybrid aspen, endeavours to shed light on the intricate mechanisms behind the control of plant development by seasonal cues, especially those related to temperature-mediated bud dormancy.

In temperate and boreal regions, perennial plants experience temperature extremes, from freezing winters to warm summers. To survive, they employ sophisticated mechanisms that detect seasonal variations and adjust their growth accordingly (Fig 1). In late summer, short days trigger the cessation of growth and protect the shoot apical meristem (SAM) and leaf primordia in a bud from winter damage. Dormancy is established to prevent early growth activation until favourable conditions return in spring. As spring arrives, dormancy is released, enabling bud break and initiating the growth cycle (Fig 1). However, the molecular mechanism underlying bud dormancy release has remained a mystery. Recent research has uncovered a novel mechanism involving the control of cell-cell communication via plasmodesmata (PDs), which are intercellular channels responsible for the trafficking of growth regulators. This dynamic regulation of PDs plays a vital role in bud dormancy.

Schematic overview about the dormancy cycle in the apex of aspen: five photos arranged in a circle with arrows inbetween llustrate how the apex looks like at the different stages during the season. Additional information describing the stage of the respective stage is added to each arrow. Fig. 1: Seasonal changes occurring in the apex of hybrid aspen during dormancy
Under long-day (LD) and warm temperature (WT) conditions in the summer, trees grow actively and stop their growth upon sensing short-days (SDs) during early autumn. SDs induce dormancy in the buds in late autumn. Chilling temperatures (LT) during the winter periods promote the release of dormancy. Relatively warmer temperature in the spring promotes the bud burst, followed by active growth again in the summer.

The identification of key genes and factors, including AGL8, FT1, GA-20 oxidase, and the callose-degrading enzyme GH17, as downstream targets of AGL8, provides a unique opportunity to unravel the relationship between gene regulation, hormonal changes, cell-cell communication (via PDs), and their combined role in temperature-mediated bud dormancy control. Based on this, the research project has below objectives:

Elucidating the role of AGL8-mediated FT1 activation in inducing the release of bud dormancy

This objective aims to investigate if FT1 is a downstream target of AGL8 and if so, uncover the significance of FT1 regulation by AGL8 in the release of bud dormancy (Fig. 2). This objective involves utilization of Chromatin Immuno-Precipitation PCR (ChIP-PCR) technique to probe the interaction between AGL8 and the FT1 promoter, which is essential for confirming AGL8 binding to the FT1 promoter. Next, AGL8-overexpressing plants carrying FT1 loss-of-function mutations (AGL8ox/ft1) will be created using CRISPR-Cas9 technology. Finally, dormancy release in the AGL8ox/ft1 double mutant and the AGL8-overexpressing parental line (AGL8ox) is compared using controlled growth conditions, simulating transitions to autumn and winter followed by bud break observation.

Determination of AGL8’s role in hormonal control of bud dormancy release in response to temperature cues

This objective delves into the role of AGL8 in regulating gibberellic acid (GA) biosynthesis, a key hormonal factor influencing bud dormancy release (Fig. 2). It involves measurement of GA levels in buds from wild-type (wt), AGL8-overexpressing (AGL8ox), and AGL8-RNAi plants after dormancy release to assess AGL8's impact on GA biosynthesis. Following that, we will introduce cDNA for GA-2 oxidase, a GA-degrading enzyme, into WT and AGL8ox hybrid aspen plants. Then we will analyse dormancy release in WT and AGL8ox lines expressing GA-2 oxidase under controlled growth conditions to determine the role of GA in low-temperature-induced dormancy release. The expected outcomes of these investigations include insights into AGL8's role as a regulator of GA biosynthesis and its significance in controlling dormancy release in response to low temperatures.

Uncovering the role of AGL8-regulated PD opening in bud dormancy release

It focuses on the role of PDs and their connection to bud dormancy (Fig. 2). We aim to understand if AGL8, a key gene identified in our research, activates GH17, an enzyme linked to the breakdown of callose, which in turn opens the PDs, enabling dormancy release by employing employ transmission electron microscopy and immunogold labelling approaches.

Scheme illustrating the key objectives of the research project: Identifying how low temperature regulates dormancy release through AGL8 FT1 activation (objective !), GA biosynthesis (objective 2) and opening of plasmodesmata (objective 3)Fig. 2: Key project objectives to elucidate the dormancy release.

In summary, this research project seeks to unravel the mysteries behind bud dormancy in trees by investigating the interactions between genes, hormones, and cell-cell communication. The findings will not only advance our understanding of how plants adapt to seasonal changes but will also contribute to the competitiveness of European research, particularly in the field of developmental adaptation to seasonal variations.  

Logo of Marie Skłodowska-Curie ActionsThis project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 101025929.